PROJECT SUMMARY Locomotion is important behavior for humans and all animals. Rhythmic movements are executed by microcircuits of the spinal cord. Setting movement goals and selecting motor programs requires higher brain planning centers that funnel inputs through the basal ganglia. Intercalated between the basal ganglia and spinal cord is a critical midbrain region dubbed the mesencephalic locomotor region (MLR), which initiates movements and controls their form and intensity. At present, we know a great deal about spinal cord and basal ganglia microcircuits, and both are well represented in the neuroscience literature. However, the MLR is much less well understood, particularly at the level of ion channels and synapses that animate MLR function. This project steps in to fill that knowledge gap. Aim 1 examines the cellular ionic mechanisms used by excitatory MLR interneurons to initiate motor programs. We hypothesize that low voltage-activated Ca2+ currents produce low-threshold spike (LTS) bursts and plateau potentials to trigger motor programs. We will test this hypothesis in adult brain slices in vitro and via Cre- dependent short-hairpin RNA (shRNA) experiments to attenuate ion channel expression in excitatory MLR interneurons in unanaesthetized freely behaving adult mice. Aim 1 will also involve next-generation deep sequencing of the transcriptome of MLR neurons. We will harness those data for this project while also releasing them into the Commons for general use among neuroscientists. Aim 2 examines the synaptic mechanisms that activate MLR neurons, which we hypothesize is disinhibition via GABAB receptors. That mechanism aligns with expectations that disinhibition via the direct path of the basal ganglia evokes motor programs via the MLR. We will test our hypothesis in adult brain slices in vitro and via Cre-dependent shRNA experiments that attenuate GABAB-GIRK channel synaptic function in unanaesthetized freely behaving adult mice. If successful, this project will explain how key midbrain microcircuits initiate locomotor behaviors at the level of ion channels and synapses, and thus bridge a major knowledge gap in motor control. Given that basal ganglia, MLR, and spinal cord circuits are ubiquitous features in the brains of all vertebrates during 500 MY of evolution, the insights we develop in a genetically and electrophysiological advantageous mammalian animal model (mouse) will be generally applicable to understanding motor control across a wide array of vertebrate animals. There may be translational significance for the treatment of movement disorders as well. The PIs of the project share complementary expertise in electrophysiology and behavioral experimentation in adult mice. The PIs have an established collaboration with considerable pilot data, which led to the specific aims and hypotheses above. Regardless of experimental outcomes, this project will elucidate the cellular and synaptic bases of MLR function and advance understanding in motor control.